Hematopoietic cell transplantation (HCT) can cure a variety of benign and malignant hematopoietic disorders, but graft-versus-host disease (GVHD) remains the primary cause of transplant-related morbidity and mortality, thereby limiting the therapeutic potential of allogeneic HCT. Donor T cells that are included in the stem cell inoculums and recognize widely distributed recipient alloantigens cause GVHD, but these T cells are also beneficial by facilitating stem cell engraftment and mediating graft-versus-leukemia (GVL) effects. Therefore, an ideal approach to prevent GVHD is to selectively inactivate alloreactive T cells specific for broadly expressed alloantigens, while sparing T cells with other specificities. A dominant mechanism of tolerance mediated by regulatory T cells (Tregs) has obvious therapeutic implications in preventing GVHD. To apply Tregs in clinical HCT, current approaches are focused on adoptive transfer of polyclonal, ex vivo-expanded Tregs to transplant recipients, before or after the HCT. However, these polyclonal Tregs are expected to have a low potency in controlling GVHD and provide non-selective immune suppression thereby negating separation of GVH and GVL reactions. We demonstrate that antigen- specific Tregs are far superior to polyclonal Tregs in the prevention of acute GVHD in pre-clinical BMT models. These preclinical studies provide rationale to support the first clinical trial in testing host-alloantigen reactive Tregs in GVHD. Although host-alloantigen reactive CD4 iTregs are highly effective in controlling GVHD, they may impair the GVL effect as well. While the project is a continuum of our current research, we will shift our focus more on alloreactive CD8 iTregs, because this is Treg subset recently discovered and because they have the potential to spare or even enhance the GVL effect. Our long-term goal is to use alloantigen-reactive Tregs to prevent GVHD while preserving the GVL effect after HCT in humans. The objective of this proposal is to test effective and translatable strategies to achieve this goal in pre-clinical murine models of allogeneic BMT. As strongly supported by our preliminary data, we hypothesize that the control of GVHD while preserving the GVL activity can be achieved by a combinational therapy with allo- reactive CD4 and CD8 iTregs, or by a singular therapy with stabilized CD8 iTregs. We plan to test our hypothesis and accomplish the objective by pursuing two Specific Aims: 1) To establish the combinational therapy with CD4 and CD8 iTregs for the control of GVHD and tumor relapse; 2) To enhance the stability and potential of CD8 iTregs in the prevention of GVHD. If the aims of this project are achieved, the acquired knowledge will shift the paradigm in validating and applying alloreactive CD8 iTregs for controlling GVHD and leukemia relapse, which will eventually benefit patients with hematological malignancies.